The level of free intracellular methotrexate and the capacity for polyglutamation of methotrexate may determine the duration of tetrahydrofolate synthesis. The relationship between methotrexate polyglutamate synthesis as a function of free or exchangeable methotrexate will be studied in the Ehrlich ascites tumor and liver cells. The possible inhibition of mammalian thymidylate synthetase from the accumulation of intracellular methotrexate-polyglutamate will be studied. An evaluation of methotrexate-polyglutamates as a determinant of methotrexate toxicity will be determined in mice treated with various combinations of methotrexate, vincristine, and thymidine. An analysis of the membrane transport characteristics of methotrexate at extracellular concentrations relevant to drug levels achieved in high-dose methotrexate regimens in the presence and absence of vincristine will be investigated. Antagonism between fluoropyrimidines and methotrexate will be assessed by monitoring the suppression of tetrahydrofolate production in the presence of free methotrexate in contrast to fluoropyrimidines, and free methotrexate plus fluoropyrimidines.